Immunohistochemistry has been used to identify various cell types in dissociated cell cultures of fetal mouse spinal cord. Neurons thought to be cholinergic; i.e., immunoreactive for choline acetyltransferase, develop this immunoreactivity after several weeks in culture and constitute a small percentage of the total neurons in these cultures. The morphologic features of the CAT-positive neurons are distinct from both GABA- and VIP-immunoreactive neurons.